Reaction products of 1H-Imidazole-1-ethanol, 4,5-dihydro-, 2-(C7-C17 odd-numbered, C17-unsatd. alkyl) derivs. and sodium hydroxide and chloroacetic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An AMES study (no E.coli and S. typhimurium TA102 strain included) with the registered substance and an AMES study with a substance analogue (performed according to OECD guideline 471) are available. Both studies are negative. The results of a chromosome aberration study (OECD 473 guideline study) shows that the substance is not clastogenic in human lymphocytes under the experimental conditions of the study, however the results indicate that the substance may have the potential to disturb mitotic processes. The substance did not induce a significant increase in the mutation frequency with and without metabolic activation in the TK mutation test system (OECD guideline 476).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 1991 - May 1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Conducted equivalent to OECD Guideline 471 and according to GLP, but the study was not conducted with E.coli and/or with S. typhimurium TA102, which have an AT base pair at the primary reversion site.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

not conducted with E.coli and/or with S. typhimurium TA102

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Based on the dose finding preliminary toxicity test, the doses in the main test were chosen as 1.3, 6.4, 32, 160 and 800 µg/plate (related to active
component of the test article).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: Water is regarded as compatible with the Ames test. The minimum required solubility for the test article of 5% (corresponding with 5000 μg/plate) was reached.

Untreated negative controls:

yes

Remarks:

distilled water

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without metabolic activation: Na-azide for TA100 and TA1535; 9-Aminoacridinehydrochloride monohydrate for TA1537 and 4-nitro-1,2-phenylene diamine for TA98. Metabolic activation by S9-mix was checked with 2-aminoanthracene for all strains.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 48 hours
DETERMINATION OF CYTOTOXICITY
- Method: the number of colonies, the number of revertants and the background growth

Evaluation criteria:

After an incubation for 48 hours at 37 degrees Celsius the colonies were counted by Artek Counter Model 982B. The validity of the automatic counts is secured by manual control counting at regular time intervals. The following criteria were used for the acceptance:
-The negative controls have to be within the the expected range as defined by literature data (Maron and Ames 1983)
-The positive controls have to show sufficient effects as defined by the laboratory's experience
-The titer determination must yield a sufficient bacterial density in the suspension

Statistics:

Not applicable

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Not reported

Remarks on result:

other: all strains/cell types tested

Conclusions:

Negative at up to cytotoxic concentration (800 µg/plate) with and without metabolic activation in the Salmonella typhimurium reverse mutation assay.This study was not conducted with E.coli and/or S. typhimurium TA102, thus not detecting certain oxidising mutagens, cross-linking agents and hydrazines.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reason / purpose for cross-reference:

read-across source

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

See section Additional information on results

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

See section Additional information on results

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING STUDY:
Moderate toxicity, as indicated by reduction in revertant numbers and thinning of the background lawn, was observed in TA1535 and TA1537 at the highest dose-level tested, both in the absence and presence of S9 metabolic activation. A more pronounced effect was observed in TA100. Reduction in revertant colonies was observed at the two highest concentrations in WP2uvrA, in the absence of S9 metabolism.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment 1: slight toxicity, as indicated by thinning of the background lawn, was observed at the highest dose level in strain TA1535, TA1537 and TA100.
Experiment 2: moderate thinning of the background lawn was observed at the highest dose-level with TA98 and WP2uvrA tester strains, while marked toxicity was observed with TA1535, TA1537 and TA100 tester strains at most of the concentrations tested.
Experiment 3: toxicity, as indicated by reduction in revertant numbers and thinning of the background lawn, was observed at the highest dose-level only in strain TA1535, TA1537 and TA100, both in the absence and presence of S9 metabolism.

PRECIPITATION CONCENTRATION: none

Conclusions:

The mutagenic potential of MIRANOL ULTRA L32 (amphoacetates C12) was assessed in Salmonella typhimurium bacterial strains and Escherichia coli , according to EC method B.13-B.14 and modified OECD guideline 471, and in compliance with Good Laboratory Practice. It was concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay. This result was read across to the registered substance.

Executive summary:

The mutagenic potential of a related member of MIRANOL ULTRA L32 (amphoacetates C12) was assessed in Salmonella typhimurium bacterial strains and Escherichia coli , according to EC method B.13-B.14 and modified OECD guideline 471, and in compliance with Good Laboratory Practice.

The histidine-requiring S. typhimurium mutants TA 1535, TA 1537, TA 98, TA 100 and tryptophan-requiring E.coli mutant WP2uvrA were used in the presence and the absence of metabolic activation (+/- S9 -mix). All determinations were made in triplicate. The first experiment was conducted using a plate-incorporation method at 5 doses levels up to 5000 µg/plate. The second and third experiment were performed using a pre-incubation method. Simultaneously negative (solvent) and positive controls were used in all experiments.

In a preliminary range-finding assay, toxicity was seen.

In the plate-incorporation assay, 4 strains were treated at dose levels 313 to 5000 µg/plate, while TA100 was treated at 156 to 2500 µg/plate. Slight toxicity was observed at the highest dose level with TA1535, TA1537 and TA100, but there was no increase in the revertant numbers in any of the strains tested.

The second assay was performed with a pre-incubation step and using the same dose-range. Moderate thinning of the background lawn was observed at the highest dose level with TA98 tester strain, while marked toxicity was observed with TA1535, TA1537 and TA100 at the 3 highest dose levels. A third experiment was then performed with these 3 strains using a lower dose-range (39.1- 625 µg/plate without S9-mix, and 78.1-1250 µg/plate only for TA1535 and TA1537 in the presence of S9 -mix).

The test material did not cause any two-fold or greater increase in the mean number of revertant colonies in the test plates, both in the absence and the presence of metabolic activation. Positive controls gave the expected increases in the number of revertants, with and without S9 mix.

Under the conditions of this study, the test material did not demonstrate any in vitro mutagenic activity in these bacterial test systems.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07-May-2010 to 13-Jul-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Principles of method if other than guideline:

The recommendations of the “International Workshop on Genotoxicity Tests Workgroup” (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007).

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

The doses of Alkylamidoamine glycinate, majority in C12 & 14 (amphoacetate) were corrected for the surfactant content (38.18%). A correction factor of 2.62 was used.

Target gene:

Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Species strain
- Type and identity of media:
-RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Dose range finding test:
Without and with S9-mix, 3 hours treatment: 100, 333, 1000, 3330 and 5000 µg/mL
Without S9-mix, 24 hours treatment: 100, 333, 1000, 3330 and 5000 µg/mL and 0.3, 1, 3, 10 and 33 µg/mL
Experiment 1 (doses selected for measurement of mutation frequency):
Without S9-mix, 3 hours treatment: 0.1, 1, 5, 10, 33, 50, 75 and 100 µg/mL
With (8% (v/v) S9-mix, 3 hours treatment: 33, 100, 125, 150, 175, 200 and 225 µg/mL
Experiment 2 (doses selected for measurement of mutation frequency):
Without S9-mix, 24 hours treatment: 0.08, 0.8, 4, 8, 17, 25, 34 and 42 µg/mL
With (12% (v/v) S9-mix, 3 hours treatment: 4, 42, 84, 125, 146, 168, 210 and 230 μg/mL
(additional concentrations were not evaluated as there was a sufficient number of analysable concentrations and toxicity at higher doses)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: RPMI 1640 culture medium
- Justification for choice of solvent/vehicle: Test compound was soluble in RPMI 1640 culture medium and this has been accepted and approved by authorities and international guidelines

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9: 15 µg/mL for the 3 hours treatment period and 5 µg/mL for the 24 hours treatment period

Positive control substance:

cyclophosphamide

Remarks:

with S9: 7.5 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Short-term treatment
With and without S9-mix: 3 hours
Prolonged treatment period
Without S9-mix: 24 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 to 12 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
- Solvent controls: Duplicate cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 9.6 x 10E5 cells plated/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)

Evaluation criteria:

The global evaluation factor (GEF) has been defined by the IWTG as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/mL

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 100 µg/mL and higher in the absence of S9, 3 hours treatment; at dose levels of 333 µg/mL and higher in the presence of S9, 3 hours treatment; at dose levels of 100 µg/mL and higher in the absence of S9, 24 hours treatment

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the absence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 84 and 90% compared to the total growth of the solvent controls after the 3 and 24 hours treatment period, respectively.

In the presence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 89 and 92% compared to the total growth of the solvent controls after the 3 hours treatment period in the first and second experiment, respectively.

In the first experiment in the presence of S9-mix (3h treatment), there were only 7 analysable concentrations obtained, due to toxicity. However these conditions were repeated in the second experiment where 8 analysable concentrations were evaluated. This slight deviation was not considered to alter the study integrity.

In the second experiment in the presence of S9-mix, the recommended toxic range of approximately 10-20% RTG was not covered in the conditions with S9-mix. But the RTG was 8%, and a mutation frequency similar to that of the next inferior dose within a very narrow dose range, and not more than 2-fold compared to the negative control. The test conditions were similar to those of the first experiment where an appropriate RTG range was obtained, except for the metabolic activation concentration which was higher in experiment 2.

Remarks on result:

other: L5178Y/TK+/-3.7.2C

Conclusions:

The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range .

Positive control chemicals, methyl methane sulfonate and cyclophosphamide induced appropriate responses.

In the absence of S9-mix, Alkylamidoamine glycinate, majority in C12 & 14 (amphoacetate) did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in a repeat experiment with modifications in the duration of treatment time.

In the presence of S9-mix, Alkylamidoamine glycinate, majority in C12 & 14 (amphoacetate) did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modifications of the S9-mix concentration added for metabolic activation.

In conclusion, Alkylamidoamine glycinate, majority in C12 & 14 (amphoacetate) is not mutagenic in the TK mutation test system under the experimental conditions described in the report.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Ty

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 29, 2010 - March 23, 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

The doses of Alkylamidoamine glycinate, majority in C12 & 14 (amphoacetate) were corrected for the surfactant content (38.18%). A correction factor of 2.62 was used.

Species / strain / cell type:

lymphocytes: human peripheral blood

Details on mammalian cell type (if applicable):

- Type and identity of media:
Blood samples
Blood samples were collected by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.

- Culture medium
Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) and 30 U/mL heparin.

- Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/mL) phytohaemagglutinin was added.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Dose range finding test:
Without and with S9-mix, 3hr exposure; 24 hr fixation: 100, 333, 1000, 3330 and 5000 µg/mL
Without S9-mix, 24/48hr exposure; 24/48 hr fixation: 100, 333, 1000, 3330 and 5000 µg/mL
First cytogenetic test (doses selected for analysis):
Without and with S9-mix, 3 h exposure time, 24 h fixation time: 100, 450 and 500 µg/ mL
Second cytogenetic test (doses selected for analysis):
Without S9-mix, 24 hr exposure; 24 hr fixation: 100, 200 and 300 µg/ mL
Without S9-mix, 48 hr exposure; 48 hr fixation: 10, 200 and 240 µg/ mL
With S9-mix, 3 hr exposure; 48 hr fixation: 100, 300 and 400 µg/ mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: RPMI 1640 medium
- Justification for choice of solvent/vehicle: Test compound was soluble in RPMI 1640 medium and this solvent has been accepted and approved by authorities and international guidelines

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9: in Hank's Balanced Salt Solution: 0.5 µg/mL for a 3 h exposure period, 0.2 µg/mL for a 24 h exposure period and 0.1 µg/mL for a 48 h exposure period

Positive control substance:

cyclophosphamide

Remarks:

with S9: in Hank's Balanced Salt Solution: 10 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration: 3 hr (with and without S9-mix), 24 and 48 hr (without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 hr

SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicates in two independent experiments

NUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of each culture was determined by counting the number of metaphases per 1000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.

Statistics:

The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/mL

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 100 µg/ml and above in the absence of S9, 3 hr treatment/24 hr fixation; at dose levels of 333 and 100 µg/ml and above in the absence of S9 for the continuous treatment of 24 and 48 hr, respectively and at dose levels of 333 µg/ml and above in the presence of S9, 3 hours treatment, 24 hours fixation

COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Appropriate toxicity was reached at the dose levels selected for scoring.

It was noted that Alkylamidoamine glycinate, majority in C12 & 14 (amphoacetate) increased the number of polyploid cells in the first cytogenetic assay both in the absence and presence of S9-mix in a dose dependent manner. In the second cytogenetic assay in the presence of S9-mix the number of polyploid cells was increased at the highest concentration tested. This may indicate that Alkylamidoamine glycinate, majority in C12 & 14 (amphoacetate) has the potential to disturb mitotic processes.

No effects of the test substance on the number of cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix.

 

Conclusions:

An in vitro chromosome aberration study was performed with the registered substance according to OECD guideline 473 and GLP principles. The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Alkylamidoamine glycinate, majority in C12 & 14 (amphoacetate) did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independent repeat experiments.

Finally, it is concluded that this test is valid and that Alkylamidoamine glycinate, majority in C12 & 14 (amphoacetate) is not clastogenic in human lymphocytes under the experimental conditions described in this report. Based on the presence of an increased number of polyploid cells in the first cytogenetic assay both in the absence and presence of S9-mix, the test item may have the potential to disturb mitotic processes.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The presence of an increased number of polyploid cells in the first cytogenetic assay both in the absence and presence of S9-mix indicated that the test item has the potential to disturb mitotic processes. Therefore an in vivo mouse bone marrow cytogenetic assay was performed. The result of the in vivo study demonstrate that the registered substance does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations in vivo.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Sep 2017 - 19 Nov 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

In vivo testing was triggered by the positive outcome of an in vitro study. The in vivo study was initiated after final approval by ECHA.

Qualifier:

according to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Version / remarks:

29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.11 (Mutagenicity - In Vivo Mammalian Bone-Marrow Chromosome Aberration Test)

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian bone marrow chromosome aberration test

Specific details on test material used for the study:

Specific gravity / density: 1.1
A correction was made for the purity/composition of the test item. The correction factor was 2.554 based on the dry-content.
Solubility in vehicle Water: Soluble
Stability in vehicle: Stability for at least 6 hours at room temperature under normal laboratory light conditions is confirmed over the concentration range 1.0 to 200 mg/mL (Charles River Project 518378). Stability for at least 7 days in the refrigerator is confirmed over the concentration range 1.0 to 200 mg/mL (Charles River Project 518378).

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

NMRI mice (SPF) were used as test system. These mice are recommended by international guidelines (e.g. OECD, EC).

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6 weeks
- Weight at study initiation: 29 - 39 g
- Assigned to test groups randomly: yes
- Fasting period before study: yes, 3 - 4 hours prior to dosing until maximum 1 hour after administration
- Housing: animals were group housed (maximum 5 animals per sex per cage) in labelled polycarbonate cages containing sterilized sawdust as bedding material. Paper bedding was provided as cage enrichment.
- Diet: free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), except prior to dosing
- Water: free access to tap-water
Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 44-60
- Air changes (per hr): 10 or more
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 11 Sep 2017 To: 13 Oct 2017

Route of administration:

oral: gavage

Vehicle:

Solvent used: water for injection
A solubility test was performed based on visual assessment. Miranol Ultra C32 was dissolved in water for injection. The dosing volume was 10 mL/kg body weight.

Details on exposure:

Miranol Ultra C32 concentrations were dosed within 3 hours after preparation.

Duration of treatment / exposure:

12 - 18 hours (three doses) and 36 - 44 hours (high dose only)

Frequency of treatment:

Single exposure

Post exposure period:

12 - 18 hours (three doses) and 36 - 44 hours (high dose only)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Remarks:

(based on dry weight)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

(based on dry weight)

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Remarks:

(based on dry weight)

No. of animals per sex per dose:

6 males (dose range finding study); 5 males (main study)

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide, 40 mg/kg bw

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

Three to five hours prior to sacrifice, animals are treated with the metaphase-arresting chemical colchicine. 12 - 18 hours (1.5 x normal cell cycle length) and 36 - 44 hours (24 hours after the first sample time) after dosing the animals are sacrificed and bone marrow cells are collected. The cells are treated with a hypotonic solution and fixed. Finally, the cell material is spread on slides and stained. Metaphase cells are examined microscopically for the presence of structural chromosome aberrations such as breaks, gaps, minutes, dicentrics and exchange figures.
The mitotic index of bone marrow metaphase slides was determined by counting the number of metaphases from at least 1000 (with a maximum deviation of 5%) cells per animal. To prevent bias, all slides were randomly coded before examination of chromosome aberrations. One hundred twenty-five to two hundred metaphase chromosome spreads per animal were examined by light microscopy for chromosome aberrations. In case the number of aberrant cells, gaps excluded, was ≥ 50 in 100 metaphases no more metaphases were examined. Only metaphases containing 40 ± 2 centromeres (chromosomes) were analyzed. Type and number of aberrations were given separately for each animal, the total number of aberrations and the number of cells with aberrations per group was calculated.

Evaluation criteria:

A bone marrow metaphase test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database and produce a statistically significant increase compared to the negative control. If (one of) the acceptability criteria are not met and the Study Director decides that this has a critical effect on the study, the test will be rejected and repeated.

Statistics:

Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) was used for statistical analysis of the data.
A test item is considered positive in the bone marrow metaphase test if all of the following criteria are met:
a) At least one of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in the frequency of cells with structural chromosome aberrations (excluding gaps) compared with the concurrent negative control
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the historical control data range.
A test item is considered negative in the bone marrow metaphase test if:
a) None of the treatment groups exhibits a statistically significant (one-sided, p < 0.05)
increase in the frequency of cells with structural chromosome aberrations (excluding
gaps) compared with the concurrent negative control.
b) There is no dose related increase when evaluated with a trend test.
c) All results are within the negative historical control data range.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- In the dose range finding test, three males and three females were dosed with 2000 mg Miranol Ultra C32/kg body weight. The animals showed no clinical signs after dosing. Since there were no differences between sexes in toxicity only male animals were used in the main study, five male animals were used in each treatment group.

RESULTS OF DEFINITIVE STUDY
- The animals of the groups treated with 2000, 1000 and 500 mg Miranol Ultra C32/kg body weight and the animals of the negative and positive control groups showed no treatment related clinical signs or mortality.
- No biologically relevant decrease in the mitotic index was observed in animals treated with Miranol Ultra C32. The inhibition of the mitotic index of the positive control was 10%.
- The scores for the number of aberrant cells (gaps included and excluded) and the number of the various types of chromosome aberrations at the various concentrations of Miranol Ultra C32 are attached.
- The number of cells with chromosome aberrations found in the animals dosed with the vehicle control was within the laboratory historical control data range. The positive control chemical produced a statistically significant increase in the frequency of aberrant cells. It was therefore concluded that the test conditions were appropriate for the detection of a clastogenic response. Miranol Ultra C32 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations, at both sampling times. No effects of Miranol Ultra C32 on the number of polyploid cells and cells with endoreduplicated chromosomes were observed.

Conclusions:

Based on the results of a mouse bone marrow cytogenetic assay was performed according to OECD/EC guidelines and GLP principles, it is concluded that Miranol Ultra C32 does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations in vivo.

Executive summary:

A mouse bone marrow cytogenetic assay was performed with Miranol Ultra C32 according to OECD/EC guidelines and GLP principles. Male mice (5/group) were exposed to 500, 1000 or 2000 mg/kg bw/day and bone marrow was sampled 12-18 (all doses, vehicle control group and positive control group (treated with cyclophosphamide) or 36-44 (highest dose only) hours after dosing. No mortality occurred, no clinical signs were noted in any of the mice. The number of cells with chromosome aberrations found in the vehicle control animals was within the laboratory historical control data range. The positive control animals treated with cyclophosphamide induced a statistically significant increase in the number of cells with chromosome aberrations, indicating that the test conditions were adequate. Miranol Ultra C32 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations, at both sampling times.

Based on these results it is concluded that Miranol Ultra C32 does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations in vivo.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

AMES studies (OECD 471):

Based on the key study (Hillmann 1991), it is concluded that the substance is negative with and without metabolic activation in the Salmonella typhimurium reverse mutation assay, under the conditions of the assay and up to the cytotoxic concentration. The mutagenic potential of a related member of the chemical category (amphoacetates C12) was assessed in Salmonella typhimurium bacterial strains and Escherichia coli, according to EC method B.13-B.14 and modified OECD guideline 471, and in compliance with GLP principles. It was concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay. These results are read-across to this substance, member of the same chemical category.

 

Chromosome aberration study (OECD 473):

The substance is not clastogenic in human lymphocytes under the experimental conditions of the study. The substance may have the potential to disturb mitotic processes.

 

Mouse Lymphoma study (OECD 476):

The substance did not induce a significant increase in the mutation frequency with and without metabolic activation in the TK mutation test system under the experimental conditions of the study.

 

In vivo bone marrow chromosome aberration Test (OECD 475):

A mouse bone marrow cytogenetic assay was performed with Miranol Ultra C32 according to OECD/EC guidelines and GLP principles. Male mice (5/group) were exposed to 500, 1000 or 2000 mg/kg bw/day and bone marrow was sampled 12-18 (all doses, vehicle control group and positive control group (treated with cyclophosphamide) or 36-44 (highest dose only) hours after dosing. No mortality occurred, no clinical signs were noted in any of the mice. The number of cells with chromosome aberrations found in the vehicle control animals was within the laboratory historical control data range. The positive control animals treated with cyclophosphamide induced a statistically significant increase in the number of cells with chromosome aberrations, indicating that the test conditions were adequate. Miranol Ultra C32 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations, at both sampling times. Based on these results it is concluded that Miranol Ultra C32 does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations in vivo.

Justification for classification or non-classification

Based on the available data, Alkylamidoamine glycinate majority C12, 14 (amphoacetate) is not classified for genotoxicity according to CLP Regulation (EC) No. 1272/2008.